Affinity and kinetics of proprotein convertase
subtilisin
⁄
kexin type 9 binding to low-density lipoprotein
receptors on HepG2 cells
Seyed A. Mousavi
1
, Knut E. Berge
1
, Trond Berg
2
and Trond P. Leren
1
1 Unit for Cardiac and Cardiovascular Genetics, Department of Medical Genetics, Oslo University Hospital Rikshospitalet, Norway
2 Department of Molecular Biosciences, University of Oslo, Norway
Keywords
association; dissociation; dissociation
constants; low-density lipoprotein receptor;
proprotein convertase subtilisin ⁄ kexin 9
Correspondence
T. P. Leren, Unit for Cardiac and
Cardiovascular Genetics, Department of
Medical Genetics, Oslo University Hospital
Rikshospitalet, P.O. Box 4950 Nydalen,
NO-0424 Oslo, Norway
Fax: +47 23075561
Tel: +47 23075552
E-mail: [email protected]
(Received 29 March 2011, revised 7 June
2011, accepted 16 June 2011)
doi:10.1111/j.1742-4658.2011.08219.x

125
I]TC-PCSK9 under pre-equilibrium
conditions was faster than under equilibrium conditions. Overall, the data
suggest that PCSK9 binding to cell surface LDLR cannot be described by
a simple bimolecular reaction. Possible interpretations that can account for
these observations are discussed.
Introduction
Proprotein convertase subtilisin ⁄ kexin type 9 (PCSK9)
is a protein secreted by the liver that was recognized as
an important regulator of cholesterol homeostasis
through its link to autosomal dominant hypercholester-
olemia [1–3]. Central to its role as a cholesterol-regula-
tory protein is the ability of PCSK9 to downregulate the
low-density lipoprotein (LDL) receptor (LDLR) [4–6].
Hepatic LDLR seems to be particularly susceptible to
this effect of PCSK9. PCSK9-mediated downregulation
of hepatic LDLR inhibits LDL uptake from plasma,
thus increasing the concentrations of LDL cholesterol
in plasma [4,7]. Besides the effect on hepatic LDLR
levels, endocytosis of PCSK9 by the liver is also
responsible for clearance of PCSK9 from the circulation
[7].
The importance of PCSK9 in maintaining choles-
terol homeostasis is clinically evident, in that PCSK9
gain-of-function mutations are associated with elevated
Abbreviations
ECD, extracellular domain; EGF-A, epidermal growth factor-like repeat A; LDL, low-density lipoprotein; LDLR, low-density lipoprotein
receptor; LPDS, lipoprotein-depleted serum; PCSK9, proprotein convertase subtilisin ⁄ kexin type 9; PCSK9-WT, wild-type proprotein
convertase subtilisin ⁄ kexin type 9; TC, tyramine cellobiose.
2938 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS

125
I]TC-PCSK9) binding
to HepG2 cells
Incubation of cells in lipoprotein-free medium increa-
sed the level of LDLR expressed on the cell surface
 1.8-fold (1.82 ± 0.10, n = 3) as compared with cells
that had been grown for 48 h in complete growth med-
ium (Fig. 1A). Under both conditions, the amount of
[
125
I]TC-PCSK9 specifically bound was linearly related
to cell density. Incubation of cells in lipoprotein-free
medium was also associated with a  1.6-fold increase
(1.64 ± 0.16, n = 3) in the binding of [
125
I]TC-PCSK9
(Fig. 1B). The binding of [
125
I]TC-PCSK9-D374Y is
presented for comparison. Approximately five times
less [
125
I]TC-PCSK9-D374Y than [
125
I]TC-PCSK9 was
needed to achieve equivalent binding (1.7 ± 0.07,
n = 3) (Fig. 1C), which is consistent with the higher
affinity of PCSK9-D374Y for LDLR at neutral pH
(see below). The extent of binding to blank wells was
similar for both ligands, and the radioactivity asso-

for cells grown in complete growth medium and LPDS-containing
Opti-MEM, respectively. The numbers of cells in wells with lower
cell numbers could not be reliably determined. The results are
means ± standard deviations of triplicate determinations from a sin-
gle experiment. Similar results were obtained in several indepen-
dent single-point binding experiments at high cell density, each
performed in duplicate. The values given in the text are the
means ± standard deviations of three independent experiments
performed at high cell density, including the results obtained with
the highest cell density in this experiment.
S. A. Mousavi et al. Characterization of the binding of PCSK9 to intact cells
FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS 2939
As the gain-of-function D374Y mutation is localized
in the LDLR-binding region of PCSK9, the enhanced
binding of [
125
I]TC-PCSK9-D374Y to HepG2 cells can
be attributed entirely to its higher affinity for LDLR.
We therefore conclude that LDLR is the main receptor
responsible for binding of [
125
I]TC-PCSK9 and
[
125
I]TC-PCSK9-D374Y to HepG2 cells.
To further demonstrate the specificity of [
125
I]TC-
PCSK9 binding, we incubated the cells for 30 min at
4 °C in the presence of different unlabeled ligands

specificity of the binding.
Estimation of binding affinity of wild-type and
two mutant variants of PCSK9
In preliminary saturation experiments, we found that it
was very difficult to achieve complete saturation curves
for [
125
I]TC-PCSK9 binding to HepG2 cells. More-
over, large amounts of unlabeled PCSK9 (wild type
and D374Y) were required to determine nonspecific
binding. These technical limitations precluded determi-
nation of equilibrium dissociation constants (K
d
) for
[
125
I]TC-PCSK9 and [
125
I]TC-PCSK9-D374Y.
In order to estimate the binding affinities of PCSK9
and PCSK9-D374Y for HepG2 cell LDLR, we incu-
bated the cells with a fixed concentration of [
125
I]
TC-labeled ligand in the presence of increasing concen-
trations of the unlabeled counterpart (Fig. 2B,C). The
abilities of unlabeled PCSK9-D374Y and PCSK9-
S127R to reduce [
125
I]TC-PCSK9 binding were also

These estimates of the relative proportions of sites are
different from those estimated by nonlinear regression
analysis. However, it has been well established that the
Scatchard method is sensitive to slight experimental
errors, making accurate estimates of the number of
binding sites from Scatchard plots difficult [22,23].
The higher-affinity and lower-affinity sites for
PCSK9 binding on HepG2 cells may be indicative of
either the presence of two subpopulations of LDLR
with different affinities for PCSK9, or negative cooper-
ativity among interacting LDLRs, although other
explanations are also possible (see below). The Hill
coefficient (the slope of Hill plot) is often used as a
measure of the extent of cooperativity, and a Hill coef-
ficient < 1.0 might suggest negative cooperativity [24].
However, the average Hill coefficient calculated for
PCSK9-WT was equal to unity (0.98 ± 0.06, n = 3),
and that obtained for PCSK9-D374Y was not signifi-
cantly different from unity (0.87 ± 0.07, n = 3) (not
shown).
Kinetic characteristics of [
125
I]TC-PCSK9 binding
to HepG2 cells
To determine whether the kinetics of binding of
PCSK9 to HepG2 cells can be described as a simple
bimolecular reaction, the kinetics of [
125
I]TC-PCSK9
and [

125
I]TC-
PCSK9-D374Y for cell surface LDLR. For both
[
125
I]TC-PCSK9 and [
125
I]TC-PCSK9-D374Y, the time
courses of binding were biphasic, and data from indi-
vidual association curves were well fitted by a two-
phase exponential association model, suggesting that
surface binding has two components, one rapid and
one slow. The half-time for association of [
125
I]TC-
PCSK9 with the rapid component, representing 35%
(± 5.3%) of specific equilibrium binding, was 6.6 min
(± 1.03 min), whereas the half-time for binding to the
slow component was 94 min (± 23 min) (n = 3). The
corresponding half-times for [
125
I]TC-PCSK9-D374Y
binding were 6.1 min (± 0.8 min) and 89 min
(± 9 min) (n = 3), respectively. The observed associa-
tion rate constants k
obs
for the rapid phase [k
obs(rapid)
]
and for the slow phase [k

125
I]TC-PCSK9 (2.5 lgÆmL
)1
) binding to
HepG2 cells ( 9.8 · 10
5
) by increasing concentrations of unlabeled
wild-type (upper curve), D374Y (lower curve) and S127R (middle
curve) variants of PCSK9. Bars the denote range of duplicate deter-
minations. For wild-type PCSK9 and PCSK9-S127R, only the upper
and lower, respectively, halves of the ranges are shown, to avoid
overlap of the error bars. The results are expressed as percentage
of control (c.p.m. in the absence of unlabeled ligand), plotted
against the concentration of unlabeled ligands. (B) Inhibition of
[
125
I]TC-PCSK9 binding to HepG2 cells (9.2–9.8 · 10
5
) by increasing
concentrations of unlabeled wild-type PCSK9. Error bars are stan-
dard deviations for data from three separate experiments [including
the one shown in (A)]. The results are expressed as a percentage
of c.p.m. in the absence of unlabeled wild-type PCSK9, plotted
against the concentration of unlabeled wild-type PCSK9. Inset: a
representative Scatchard plot of the competition for [
125
I]TC-PCSK9
binding by unlabeled wild-type PCSK9. (C) Inhibition of [
125
I]TC-

amount of [
125
I]TC-PCSK9 that remained bound after
6 h was about 40%. Approximately 20% of the bound
[
125
I]TC-PCSK9-D374Y dissociated rapidly, with a
half-time of 21 min (± 3.7 min), and the remaining
bound [
125
I]TC-PCSK9-D374Y dissociated more
slowly, with a half-time of 297 min (± 25 min)
(n = 3). The fraction of [
125
I]TC-PCSK9-D374Y that
remained bound after 6 h was about 45%. The dissoci-
ation rate constants [k
off
] for the rapid phase [k
off(rapid)
]
and for the slow phase [k
off(slow)
] are shown in Table 2.
Taken together with the association data, these
results suggest that the increased affinity of PCSK9-
D374Y for cell surface LDLR is mainly determined by
the rate of association. It should be noted that quanti-
tative analysis of the more rapid phase of association
requires measurement of the binding on time scales of

D374Y, the half-time of the rapid phase of [
125
I]TC-
PCSK9-D374Y dissociation was reduced from 21 to
19 min, and the half-time of the slow phase was reduced
from 297 to 179 min (Fig. 4B). These results suggest
that the reason for ligand remaining bound to cells after
6 h is not irreversibility of the binding.
Dissociation of [
125
I]TC-PCSK9 at low pH
As the affinity of PCSK9 for the ECD or the EGF-A
domain of LDLR is known to increase at acidic pH
Table 1. Parameters obtained from binding-inhibition experiments. Best-fit values for IC
50
were derived from nonlinear regression analysis.
K
d
values were derived separately from Scatchard plots. High and low represent affinities of binding sites for unlabeled ligands. Values in
parentheses indicate the number of experiments performed. The data from each experiment were analyzed separately, and mean val-
ues ± standard deviations were calculated from these values.
Labeled PCSK9 Competitive ligand
IC
50
(nM) K
d
(nM)
High Low High Low
[
125

centage of total radioactivity added. (B) Binding presented as the
amount of [
125
I]TC-labeled ligand specifically bound. Error bars are
standard deviations for data from three separate experiments. The
binding data were normalized for cell number (per 10
6
cells). The
curves were fitted with the two-phase exponential association
model.
Characterization of the binding of PCSK9 to intact cells S. A. Mousavi et al.
2942 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS
[15,25], it is believed that, subsequent to internalization
of the PCSK9–LDLR complex, LDLR is diverted to a
degradation pathway, owing to persistence of the com-
plex at endosomal pH [15]. However, the rates of dis-
sociation of PCSK9 that has previously bound to
LDLR at neutral pH have not been determined under
different pH conditions to test this hypothesis. The
effect of pH on the dissociation of [
125
I]TC-PCSK9
was measured at pH 6.2 (to mimic the early endosomal
pH). As shown in Fig. 4A, low pH did indeed mark-
edly reduce the dissociation of [
125
I]TC-PCSK9 from
cells. Dissociation occurred as a monophasic process
with a rate constant [k
off

tion time was investigated to determine whether the
proportions of the two kinetic components correspond
to the presence of two distinct receptor sites ⁄ states
with different and fixed affinities. The prediction of
this mechanism is that the proportions of the two
components will be constant ( 3 : 1 for [
125
I]TC-
PCSK9 and  4 : 1 for [
125
I]TC-PCSK9-D374Y) and
independent of the length of association time. This
prediction was tested by comparing the dissociation
rate for binding under the pre-equilibrium conditions
(60 min) and equilibrium conditions (240 min). As can
be seen in Fig. 5A, dissociation of [
125
I]TC-PCSK9
was biphasic at both association times. Approximately
42% and 22% of dissociation occurred in the rapid
phase after short and long incubation times, respec-
tively. The dependence of the kinetics of [
125
I]TC-
PCSK9-D374Y dissociation on the length of binding
time was also examined. Again, dissociation from the
rapid component was found to be faster at 60 min
than at 240 min (36% versus 19% under equilibrium
conditions) (Fig. 5B).
These data suggest that there is a fraction of rapidly

decay phase model. k
off(rapid)
and k
off(slow)
are the dissociation rate constants for the rapid and slow dissociation components, respectively.
k
obs
values were obtained by fitting the time course data with a two-exponential association model. k
obs(rapid)
and k
obs(slow)
are the observed
association rate constants for the rapid and slow association phases, respectively. The values for the constants are means ± standard devia-
tions of three experiments. The data from each experiment were analyzed separately. The concentrations used are those described in the
text.
Labeled ligand
k
off
(min
)1
) k
obs
(min
)1
)
k
off(rapid)
k
off(slow)
k

states with affinities for PCSK9 that differ approxi-
mately five-fold. An apparently good fit of nonlinear
regression analysis of binding data to a two-site model
was also obtained, suggesting that the equilibrium
binding of PCSK9 to cell surface LDLR is not a sim-
ple bimolecular reaction (see Doc. S1, model A).
One possibility that could explain the observed cur-
vilinear Scatchard plots is that unlabeled and labeled
ligands have different affinities for the receptors [32].
However, this seems to be less likely, as the presence
of two classes of PCSK9 binding site were also
observed in kinetic experiments where only [
125
I]TC-
labeled ligands were employed. Moreover, we believe
that the [
125
I]TC-labeling method, in contrast to the
direct
125
I-labeling method, does not appreciably alter
the binding properties of PCSK9, as [
125
I]TC-PCSK9-
Fig. 5. Dissociation kinetics of [
125
I]TC-PCSK9 (A) and [
125
I]TC-
PCSK9-D374Y (B) as a function of time of association: HepG2 cells

Error bars are standard deviations for data from three separate
experiments. Asterisks indicate error bars representing mean ±
one-half the range from two separate experiments. Dissociation in
the absence (control) and presence of unlabeled ligand was best
described by a two-exponential decay phase model, whereas disso-
ciation at low pH was best described by a one-exponential decay
phase model. Final concentrations of unlabeled ligands in the disso-
ciation medium were 120 lgÆmL
)1
(wild-type PCSK9) and
30 lgÆmL
)1
(PCSK9-D374Y). If the K
d
values estimated here are
assumed, then about 70% of the high-affinity sites and about 12%
of the low-affinity sites are expected to be occupied at the concen-
trations of unlabeled ligands used.
Characterization of the binding of PCSK9 to intact cells S. A. Mousavi et al.
2944 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS
D374Y consistently displayed a much higher affinity
for HepG2 cell surface receptors than did [
125
I]TC-
PCSK9. Binding to sites other than LDLR, such as
LDLR related protein 1 (LRP1) [4], may also be
responsible for the observed curvature. However, we
consider this possibility to be less likely, because LRP1
has been shown to be not regulated by cellular choles-
terol levels [33], and therefore cannot account for the

or 20-fold lower (6 nm) [25] than the K
d
estimated
here. The apparent K
d
of PCSK9-S127R binding to
the high-affinity LDLR site (548 nm), as measured by
its ability to inhibit [
125
I]TC-PCSK9 binding to HepG2
cells, was slightly lower (i.e. slightly higher affinity)
than that for wild-type PCSK9, and is comparable to
the 648 nm K
d
obtained in a Biacore study [19]. The
K
d
values derived for the lower-affinity class of sites
are shown in Table 1. In this context, it is worth men-
tioning that the existence of high-affinity (32 nm) and
low-affinity (86 nm) states has previously been demon-
strated for binding of PCSK9-S127R to the ECD of
LDLR at pH 7.5 [25]. This study also found high-
affinity (1 nm) and low-affinity (42 nm) binding states
for the interaction between wild-type PCSK9 and the
ECD of LDLR at pH 5.4, whereas PCSK9-D374Y
binds with only one affinity (K
d
=6nm) at pH 7.5
and with a slightly higher affinity (K

model, dimeric and monomeric receptor states bind
PCSK9 with equal affinity, but they differ in their con-
version rates, i.e. rate constants governing the confor-
mational change that leads to the second binding step.
Thus, the rapid phase of PCSK9 association could rep-
resent binding of PCSK9 to dimeric receptors that
release bound ligand slowly because they convert rap-
idly. The slow phase of association could represent
binding to monomeric receptors that release bound
ligand rapidly because they convert slowly. The pro-
posed model is supported by the finding that a signifi-
cant proportion of LDLRs in the plasma membrane
pre-exist as noncovalent dimers (or higher oligomers)
in coated pits [36–38] or even outside coated pits [39],
and by the recent demonstration that PCSK9 can also
bind, via its C-terminal domain, to the LDL-binding
domain of LDLR [18].
The molecular basis of the enhanced rate of dissoci-
ation observed in the presence of unlabeled ligand is
unclear. This phenomenon has often, but not always,
been interpreted as indicative of the presence of nega-
tive cooperativity, i.e. a decrease in affinity with
increasing site occupancy [40]. At the present time, a
mechanistic explanation of negative cooperativity, if
present, in this system would be difficult, although
negative cooperativity among partially occupied
dimeric receptors or between two binding sites on a
monomeric divalent receptor [41] cannot be excluded.
It should be noted that, given the small amount of
[

125
I-TC-PCSK9
association and dissociation rates at 37 °C is difficult
to obtain, because, as discussed in Results, in contrast
to many ligand–receptor systems, acid wash does not
favor dissociation of [
125
I]TC-PCSK9 from the plasma
membrane. We are currently trying to develop a wash
method that can effectively remove cell surface-bound
[
125
I]TC-PCSK9.
Experimental procedures
Materials
Culture media and antibiotics, l-glutamine and nonessential
amino acids were from Gibco BRL (Invitrogen, Carlsbad,
CA, USA). Antibody against LDLR was from RDI
Research Diagnostic (Concord, MA, USA). BSA and fetal
bovine serum were from Sigma Aldrich (St Louis, MO,
USA). Na
125
I was purchased from PerkinElmer (Waltham,
MA, USA). IodoGen-precoated tubes were from Pierce
Biotechnology (Rockford, IL, USA). All other chemicals
and reagents were obtained from Sigma Aldrich unless
otherwise specified.
Protein expression and purification
PCSK9-D374Y and PCSK9-S127R are two naturally occur-
ring gain-of-function mutants of PCSK9 that cause severe

properties is unclear, but the results suggest that the bind-
ing of [
125
I]PCSK9 to a nonreceptor site is particularly
enhanced by the radio-iodination of a tyrosine(s). It should
be noted that the EGF-A-binding region of PCSK9 con-
tains no tyrosines (or lysines).
Purified wild-type PCSK9 was covalently coupled to
[
125
I]TC by the method of Pittman et al. [45], with modifi-
cations as described previously [46]. Briefly, [
125
I]TC was
prepared by reacting TC (6 lLof10mm solution in
NaCl ⁄ P
i
) with Na
125
I (1.0 mCi) in IodoGen-precoated
tubes (Pierce) for 30–40 min at room temperature, followed
by transfer to a tube containing cyanuric chloride (6 lLof
10 mM solution in acetonitrile) and potassium iodide (6 lL
of 0.1 m solution) for 3 min. The activated [
125
I]TC adduct
was then incubated with wild-type PCSK9 (300–400 lgin
200 lL of carbonate buffer containing 0.5 mm CaCl
2
,

NaCl ⁄ P
i
containing 0.5 mm CaCl
2
, pH 7.4) was incubated
with Na
125
I (0.3 mCi) in IodoGen-precoated tubes for 10–
15 min at room temperature. The reaction was stopped by
transfer to a tube containing 0.9 mL of NaCl ⁄ P
i
containing
0.5 mm CaCl
2
(pH 7.4). Free
125
I was removed by gel filtra-
tion as described above. Goat anti-(rabbit IgG) was radio-
labeled with Na
125
I as described for direct iodination of
PCSK9, except that CaCl
2
was omitted.
Cell culture, buffers, and cell treatments
HepG2 cells (European Collection of Cell Cultures, Porton
Down, UK) were routinely cultured in collagen-coated
75-cm
2
tissue culture flasks (BD Biosciences, San Diego,

blue dye. At the end of each experiment, two wells were
treated with trypsin and the average cell number was deter-
mined with a hemocytometer. All experiments were per-
formed on several different HepG2 cell batches and, with
the exception of PCSK9-S127R (one preparation), at least
three different wild-type PCSK9 and PCSK9-D374Y prepa-
rations and five different preparations of [
125
I]TC-labeled
proteins were used.
Determination of specific binding of
[
125
I]TC-PCSK9 to HepG2 cells
Varying numbers of HepG2 cells (70 000, 140 000 and
280 000) were seeded in 12-well plates and incubated either
for 48 h in complete growth medium or for 24 h in com-
plete growth medium, and then for 26 h in 3% LPDS-con-
taining Opti-MEM. In experiments with cells grown in
complete growth medium for 48 h, cells were incubated in
lipoprotein-free medium for 2 h at 37 °C before the start of
the experiments, to allow internalization of cell surface-
bound LDL. After 50 h of incubation at 37 °C, cells were
washed once with 1 mL of DMEM and incubated in the
same medium for 15 min at 4 °C. The medium was then
removed, and triplicate wells were incubated with 0.5 mL
of incubation medium containing 10 lgÆmL
)1
of antibody
against LDLR for 90 min at 4 °C. The cells were then

point binding assays at high initial cell density.
Binding-inhibition experiments
Cells grown in 12-well plates were washed once with 1 mL
of DMEM and incubated in the same medium for at least
15 min at 4 °C. The medium was removed, and cells were
incubated with increasing concentrations of unlabeled (seri-
ally diluted two-fold) proteins in a total volume of 480 lL
at 4 °C for 15 min. Labeled ligand was added in a small
volume (20 lL) and the cells were incubated at 4 °C for
4 h. At the end of incubation, the cells were washed once
with 1 mL of cold wash buffer and three times with 1 mL
of wash buffer without BSA. The wash procedure took
 4 min per plate. After washing, cells were solubilized and
radioactivity was measured as described above.
Kinetic association experiments
Cells grown in 12-well plates were washed as described
above. The medium was removed, and the cells were incu-
bated with 0.5 mL of binding medium containing [
125
I]TC-
PCSK9 ( 5 lgÆmL
)1
,70nm)or[
125
I]TC-PCSK9-D374Y
( 1 lgÆmL
)1
,14nm). At various times, cells were washed
four times and solubilized, and radioactivity was measured
as previously described. A 200-fold excess of unlabeled

I-TC-PCSK9 (and [
125
I]TC-PCSK9-D374Y)
molecules remained intact and were still on the cell surface.
Data analysis
All binding and kinetic data were fitted by nonlinear regres-
sion with prism 5 (GraphPad Software, CA, USA). For
inhibition-binding curves, the raw data were analyzed
according to one-site-fit and two-site-fit log
IC50
models. The
data were also analyzed according to the Scatchard method
[47]. Linear regression analyses of binding data gave dissoci-
ation constants (K
d
), calculated from the reciprocal of the
slopes. Association data were fitted by either the one-phase
exponential association equation Y = Y
0
+ (plateau )
Y
0
)*[1 ) exp() Kx)] or the two-phase exponential associa-
tion equation Y = Y
0
+ SpanFast*[1 ) exp() KFast*
X)] + SpanSlow*[1 ) exp() K
Slow
X)]. Dissociation data were
fitted by either the one-phase exponential decay equation

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Supporting information
The following supplementary material is available:
Doc. S1. Models.